Inactivated Bacterial Cell Formulation

ABSTRACT

The invention relates to the use of lactic acid-producing bacteria to boost the immune system.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 15/268,276, filedSep. 16, 2016, which is a continuation of U.S. Ser. No. 14/949,362,filed on Nov. 23, 2015, now U.S. Pat. No. 9,446,111, issued on Sep. 20,2016, which is a continuation of U.S. Ser. No. 14/064,863, filed on Oct.28, 2013, now U.S. Pat. No. 9,192,659, issued on Nov. 24, 2015, which isa continuation of U.S. Ser. No. 12/770,457, filed on Apr. 29, 2010, nowU.S. Pat. No. 8,568,743, issued on Oct. 29, 2013, which claims thebenefit of priority to U.S. Provisional Application No. 61/285,255,filed on Dec. 10, 2009 and U.S. Provisional Application No. 61/214,876,filed on Apr. 29, 2009, each of which is herein incorporated byreference in their entirety.

FIELD OF THE INVENTION

The invention relates to the use of viable and non-viable bacteria toboost the immune system.

BACKGROUND OF THE INVENTION

The gastrointestinal microflora plays a number of vital roles inmaintaining gastrointestinal tract function and overall physiologicalhealth. The growth and metabolism of the many individual bacterialspecies inhabiting the gastrointestinal tract depend primarily upon thesubstrates available to them, most of which are derived from the diet.See, e.g., Gibson G. R. et al., 1995 Gastroenterology 106: 975-982;Christl, S. U. et al., 1992 Gut 33: 1234-1238. These findings have ledto attempts to modify the composition and metabolic activities of thebacterial community through diet, primarily with probiotics, which arelive microbial food supplements.

Probiotic organisms are non-pathogenic, non-toxigenic, retain viabilityduring storage, and typically survive passage through the stomach andsmall intestine. Since probiotics do not generally permanently colonizethe host, they need to be ingested regularly for any health promotingproperties to persist.

SUMMARY OF THE INVENTION

The invention describes the use of lactic acid-producing bacteria ornon-viable fragments or products thereof to boost the immune system.Specifically, the administration of Bacillus coagulans, purified cellwall components of Bacillus coagulans, or culture supernatants ofBacillus coagulans increases the immune system's ability to fightpathogens. Cell wall components and/or culture supernatants are usefulin products where conditions are not optimal for long-term vegetativecell viability, e.g., shelf stable beverages or food compositions.Alternatively, inactivated/dead Bacillus coagulans, e.g., heat killedBacillus coagulans is administered to boost the immune system. Themethods optionally include administration of purified viable Bacilluscoagulans vegetative cells and/or spores to boost the immune system.

Accordingly, a method of enhancing or boosting an immune response to amicrobial pathogen is carried out by identifying a subject infected witha microbial pathogen and administering to the subject a compositioncomprising a viable Bacillus coagulans bacterium, a non-viable fragmentof the bacterium, or a non-viable extracellular product of thebacterium. The bacteria, fragments, or products are administered in anamount that enhances the immune response of the subject to the pathogenwith which the subject is infected. Preferably, the bacteria, fragments,or products are purified or fractionated from other bacteria or othercomponents of the bacteria (in the case of fragments, e.g., cell wallfragments or secreted products).

Purified and/or isolated Bacillus coagulans is particularly useful as aprobiotic in the methods and compositions described herein. By“purified” or “substantially purified” is meant a Bacillus coagulansbacterium, a non-viable fragment of the bacterium, or a non-viableextracellular product of the bacterium that is substantially free ofcontaminating microorganisms or other macromolecules, e.g.,polysaccharides, nucleic acids, or proteins. A purified preparationcontains at least 75%, 85%, 95% or 100% of the desired composition andis substantially free of other sub-cellular components such ascytoplasmic organelles. For example, a bacterial cell wall fraction isat least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% cell wall fragments.Such a preparation is sustainably free of cytoplasm intracellularorganelles and secreted bacterial products.

In one aspect, the microbial pathogen is a bacterium or virus such as apathogen that causes a respiratory infection. For example, the pathogencomprises an influenza virus such as a human, avian, or swine influenzavirus or combination thereof. Other viral pathogens include adenovirus.

The compositions of the invention comprise an immune-enhancing amount ofa viable Bacillus coagulans bacterium, a non-viable fragment of Bacilluscoagulans bacterium, or a non-viable extracellular product of Bacilluscoagulans bacterium (e.g., a supernatant of a Bacillus coagulansbacterium). Enhancement of the immune response comprises an increase incytokine (e.g., interleukin-2 (IL-2), IL-4, IL-6, IL-10, tumor necrosisfactor-α (TNF-α), and interferon-γ (IFN-γ) production or an increase inimmune cell migration to an infection site. Immune enhancement alsoincludes boosting the immune system by increasing cytokine production,activating the immune surveillance aspect of polymorphonuclearleukocytes (PMN), increasing immune cell chemotaxis, activating NKcells, and/or increasing monocyte phagocytosis. Specifically, thecompositions of the invention increase the chemotactic abilities andphagocytic abilities of PMNs. The compositions of the invention alsoincrease the expression of CD69 on NK cells.

In one aspect, an immune-enhancing amount of Bacillus coagulans,Bacillus coagulans supernatant, or Bacillus coagulans cell wallcomponents is about 0.1 mg to about 10 grams, e.g., about 1 mg to about10 grams, about 10 mg to about 5 grams; about 100 mg to about 1 gram; orabout 200 mg to about 1 gram.

Also within the invention are compositions suitable for human ingestion,such as a composition comprising a purified cell wall of a Bacilluscoagulans bacterium or a composition comprising a dried or lyophilizedsecreted product or mixture of secreted products of Bacillus coagulans.Exemplary formulations include a pill, capsule, or suspension.

Exemplary bacterial species for the compositions and methods describedherein include Bacillus coagulans, e.g., Bacillus coagulans hammer,preferably Bacillus coagulans hammer strain Accession No. ATCC 31284, orone or more strains derived from Bacillus coagulans hammer strainAccession No. ATCC 31284 (e.g., ATCC Numbers: GBI-20 (GB-20), ATCCDesignation Number PTA-6085; GBI-30 (GB-30/Ganeden BC³⁰™/BC³⁰), ATCCDesignation Number PTA-6086; and GBI-40 (GB-40), ATCC Designation NumberPTA-6087; see, U.S. Pat. No. 6,849,256 to Farmer). Preferably, theBacillus coagulans comprises GBI-30 (BC³⁰), or any strain of theorganism described in U.S. Ser. No. 11/706,642, hereby incorporated byreference.

The Bacillus coagulans Hammer strains of the invention arenon-pathogenic and generally regarded as safe for use in human nutrition(i.e., GRAS classification) by the U.S. Federal Drug Administration(FDA) and the U.S. Department of Agriculture (USDA), and by thoseskilled in the art. Furthermore, the Bacillus coagulans Hammer strainsof the invention germinate at or below human body temperature, renderingthem useful as probiotics. Many Bacillus coagulans strains outside theHammer group have mostly industrial applications, little or nonutritional benefit, and environmental contaminants that have not beenevaluated for safety. Moreover, many other non-Hammer strains ofBacillus coagulans grow optimally at temperatures that exceed human bodytemperature and, thus, do not germinate efficiently in the human body.Such strains are less or not suitable as probiotics for humanconsumption.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims. Unless otherwise defined, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Althoughmethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present invention,suitable methods and materials are described below. All publications,patent applications, patents, Genbank/NCBI accession numbers, and otherreferences mentioned herein are incorporated by reference in theirentirety. In the case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a line graph demonstrating the percent inhibition ofspontaneous reactive oxygen species (ROS) formation afterpolymorphonuclear leukocyte (PMN) exposure to either Bacillus coagulanssupernatant (BC1) or Bacillus coagulans cell wall components (BC2),compared to baseline results.

FIG. 2 is a line graph illustrating the percent inhibition ofH₂O₂-induced ROS formation compared to baseline results.

FIG. 3 is a schematic representation of how PMN migration begins in theblood stream and moves into the tissue via transwell migration plates.

FIG. 4 is a line graph demonstrating random migration showing themigratory patterns of PMN's treated with either Bacillus coagulanssupernatant (BC1) or Bacillus coagulans cell wall components (BC2).

FIG. 5 is a line graph illustrating bacterial peptide formyl-Met-Leu-Phe(f-MLP)-directed migration showing the migratory patterns of PMS'streated with either Bacillus coagulans supernatant (BC1) or Bacilluscoagulans cell wall components (BC2).

FIG. 6A is a line graph demonstrating interleukin-8 (IL-8)-directedmigration showing the migratory patterns of PMN's treated with eitherBacillus coagulans supernatant (BC1) or Bacillus coagulans cell wallcomponents (BC2). FIG. 6B is a line graph depicting IL-8 directedmigration of Bacillus coagulans supernatant (BC1) and Bacillus coagulanscell wall components (BC2)-treated PMN cells.

FIG. 7A is a line graph illustrating leukotriene B4 (LTB4)-directedmigration showing the migratory patterns of PMN's treated with eitherBacillus coagulans supernatant (BC1) or Bacillus coagulans cell wallcomponents (BC2). FIGS. 7B-7C are a series of bar charts illustratingLTB4-directed migration.

FIG. 8 is a bar graph demonstrating synergistic random migration showingthe migratory patterns of PMN's exposed to either Bacillus coagulanssupernatant (BC1) or Bacillus coagulans cell wall components (BC2)acting as a chemoattractant in the bottom chamber of the trans-wellmigration plate.

FIG. 9 is a bar chart illustrating synergistic f-MLP-directed migrationshowing the migratory patterns of PMN's exposed to either Bacilluscoagulans supernatant (BC1) or Bacillus coagulans cell wall components(BC2) acting as a chemoattractant in the bottom chamber of thetrans-well migration plate.

FIG. 10 is a bar graph demonstrating the synergistic IL-8-directedmigration showing the migratory patterns of PMN's exposed to eitherBacillus coagulans supernatant (BC1) or Bacillus coagulans cell wallcomponents (BC2) acting as a chemoattractant in the bottom chamber ofthe trans-well migration plate.

FIG. 11 is a bar chart demonstrating the synergistic LTB4-directedmigration showing the migratory patterns of PMN's exposed to eitherBacillus coagulans supernatant (BC1) or Bacillus coagulans cell wallcomponents (BC2) acting as a chemoattractant in the bottom chamber ofthe trans-well migration plate.

FIG. 12 is a bar graph illustrating the monocyte phagocytosis asmeasured by how well the monocyte can ingest green carboxylatefluorspheres.

FIG. 13 is a bar chart demonstrating PMN phagocytosis as measured by howwell the monocyte can ingest green carboxylate fluorspheres.

FIG. 14 is a line graph showing cluster of differentiation 69 (CD69)expression of natural killer (NK) cells (analysis generated by measuringthe mean fluorescence intensity (MFI) of CD69).

FIG. 15 is a line graph showing CD25 expression of NKT cells (analysisgenerated by measuring the MFI of CD25).

FIG. 16 is a line graph demonstrating CD107a expression of NK cells(analysis generated by measuring the MFI of CD107a).

FIG. 17 is a line graph illustrating the results of lymphocytes thatwere pre-treated with either Bacillus coagulans supernatant (BC1) orBacillus coagulans cell wall components (BC2) and then exposed to themitogen phytohemagglutinin (PHA).

FIG. 18 is a line graph demonstrating the results of lymphocytes thatwere pre-treated with either Bacillus coagulans supernatant (BC1) orBacillus coagulans cell wall components (BC2) and then exposed to thepokeweed mitogen (PWM).

FIGS. 19A and 19B are line graphs showing the cytokine production oflymphocytes pre-treated with Bacillus coagulans supernatant (BC1).

FIGS. 20A and 20B are line graphs illustrating the cytokine productionof lymphocytes pre-treated with Bacillus coagulans cell wall components(BC2).

FIG. 21 is a bar chart showing the results of lymphocytes that werepre-treated with either Bacillus coagulans supernatant (BC1) or Bacilluscoagulans cell wall components (BC2), and then exposed to no mitogen,PHA, or PWM. This graph represents relative levels of the cytokine IL-2present in the supernatant of 5 day lymphocyte cultures. Untreated (UT).

FIG. 22 is a bar graph illustrating the results of lymphocytes that werepre-treated with either Bacillus coagulans supernatant (BC1) or Bacilluscoagulans cell wall components (BC2) and then exposed to no mitogen,PHA, or PWM. This graph represents relative levels of the cytokine IL-4present in the supernatant of 5 day lymphocyte cultures.

FIG. 23 is a bar chart demonstrating the results of lymphocytespre-treated with either Bacillus coagulans supernatant (BC1) or Bacilluscoagulans cell wall components (BC2), and then exposed to no mitogen,PHA, or PWM. This graph represents relative levels of the cytokine IL-6present in the supernatant of 5 day lymphocyte cultures.

FIG. 24 is a bar graph illustrating the results of lymphocytes that werepre-treated with either Bacillus coagulans supernatant (BC1) or Bacilluscoagulans cell wall components (BC2), and then exposed to no mitogen,PHA, or PWM. This graph represents the relative levels of the cytokineIL-10 present in the supernatant of 5 day lymphocyte cultures.

FIGS. 25A and 25B shows a series of bar charts showing the results oflymphocytes that were pre-treated with either Bacillus coagulanssupernatant (BC1) or Bacillus coagulans cell wall components (BC2), andthen exposed to no mitogen, PHA, or PWM. This graph represents therelative levels of the cytokine TNF-α present in the supernatant of 5day lymphocyte cultures.

FIGS. 26A and 26B show a series of bar charts showing the results oflymphocytes that were pre-treated with either Bacillus coagulanssupernatant (BC1) or Bacillus coagulans cell wall components (BC2), andthen exposed to no mitogen, PHA, or PWM. This graph represents relativelevels of the cytokine IFN-γ present in the supernatant of 5 daylymphocyte cultures.

FIG. 27 is a schematic of a cell-based antioxidant protection inerythrocytes (CAP-e) representation of how a natural product gets intothe cell. A dye is used to express fluorescence representing oxidativestress.

FIG. 28 is a line graph showing CAPe results for Bacillus coagulanssupernatant being tested in parallel on fresh and aged cells.

FIG. 29 is a line graph showing CAPe results for Bacillus coagulans cellwall being tested in parallel on fresh and aged cells.

FIG. 30A is a photomicrograph depicting an untreated PMN cell that hasengaged in phagocytosis. The green beads are carboxylated fluorospheresthat mimic bacterial particles.

FIG. 30B is a photomicrograph depicting a PMN cell treated with Bacilluscoagulans cell wall components (BC2).

FIG. 31 is an illustration of a typical protein gel electrophoresismethod.

FIG. 32 is a photograph depicting the results of a gel electrophoresisexperiment with Bacillus coagulans supernatant and cell wall fractions.

FIG. 33 is a schematic representation of how PMN migration begins in theblood stream and moves into the tissue via transwell migration plates.

DETAILED DESCRIPTION

The present invention is directed to the discovery that non-pathogeniclactic acid-producing bacteria (i.e., “lactic acid bacteria”), such asBacillus coagulans, are useful in boosting the immune system, i.e.,increasing the level of activation of immune cells. Bacillus coagulansvegetative cells and/or spores are used or inactivated/dead Bacilluscoagulans are used, e.g., heat killed Bacillus coagulans. For example,the administration of cell wall components or culture supernatants ofBacillus coagulans boosts the immune system by increasing cytokineproduction, activating the immune surveillance aspect ofpolymorphonuclear leukocytes (PMN), increasing immune cell chemotaxis,activating natural killer (NK) cells, and increasing monocytephagocytosis.

Probiotic Lactic Acid-Producing Bacteria

A probiotic lactic acid-producing bacterium suitable for use in thedescribed methods and compositions produces acid and is non-pathogenic.Bacterial species include Bacillus coagulans, e.g., Bacillus coagulanshammer, preferably Bacillus coagulans hammer strain Accession No. ATCC31284, or one or more strains derived from Bacillus coagulans hammerstrain Accession No. ATCC 31284 (e.g., ATCC Numbers: GBI-20, ATCCDesignation Number PTA-6085; GBI-30 or BC³⁰, ATCC Designation NumberPTA-6086; and GBI-40, ATCC Designation Number PTA-6087; see U.S. Pat.No. 6,849,256 to Farmer).

Viable Bacillus coagulans vegetative cells or spores, Bacillus coagulanssupernatant, and Bacillus coagulans cell wall components are useful inthe present invention. Purified and/or isolated Bacillus coagulansvegetative cells or spores, Bacillus coagulans supernatant or Bacilluscoagulans cell wall components are particularly useful as a probiotic inthe compositions described herein. By “purified” or “substantiallypurified” is meant Bacillus coagulans vegetative cells or spores,Bacillus coagulans supernatant, or Bacillus coagulans cell wellcomponents that are substantially free of contaminating microorganismsor other macromolecules, e.g., polysaccharides, nucleic acids, orproteins. A purified preparation contains at least 75%, 85%, 95% orabout 100% of the desired composition and is substantially free of othersub-cellular components such as cytoplasmic organelles. For example, abacterial cell wall fraction is at least 75%, 80%, 85%, 90%, 95%, 98%,99%, or about 100% cell wall components. Such a preparation issustainably free of cytoplasm intracellular organelles and secretedbacterial products.

The compositions include Bacillus coagulans vegetative cells or spores,Bacillus coagulans supernatant, or Bacillus coagulans cell wallcomponents in the form of a powder, a dried cell mass, a stabilizedpaste, or a stabilized gel. Bacillus coagulans vegetative cells orspores, Bacillus coagulans cell wall and/or Bacillus coagulans culturesupernatant is used in the methods described herein for boosting theimmune system. Optionally, the Bacillus coagulans cell wall and/orBacillus coagulans culture supernatant is dried and reconstituted inwater or other aqueous solution before use.

Because Bacillus spores are heat and pressure-resistant and can bestored as a dry powder, they are particularly useful for formulationinto and manufacture of products such as the various compositionsdescribed herein. A Bacillus species is well suited for the presentinvention, particularly species having the ability to form spores whichare relatively resistant to heat and other conditions, making them idealfor storage (shelf-life) in product formulations.

Optionally, the compositions comprise Bacillus coagulans vegetativecells and/or spores. The cells/spores are formulated in a variety ofcompositions suited for use in an immune-boosting composition. In oneaspect, the bacterium is present as a mixture of spores and vegetativecells. In another aspect, the bacterium is present as at least 90%spores, e.g., 95%, 98%, or 99% spores. Optionally, prior to addition tothe compositions of the invention, the Bacillus coagulans cells arecultured in liquid in the absence of or with limited quantities of afood source to induce sporulation. In another aspect, heat gun spraydrying kills about 50%, about 75%, about 90%, about 95%, or about 99% ofvegetative cells prior to addition to the compositions of the invention.In one aspect, at least about 5%-25% of the bacteria in the compositionare viable, e.g., at least about 25%-50%; at least about 50%-75%; or atleast about 75%-99% of the bacteria are viable. In another aspect, thecomposition comprises at least about 1×10⁶ to 1×10⁷; at least about1×10⁷ to 1×10⁸; or at least about 1×10⁸ to 1×10⁹ viable bacteria.

The invention also provides Bacillus coagulans cell wall and/or Bacilluscoagulans culture supernatant for use in the methods described hereinfor boosting the immune system. In one aspect, Bacillus coagulansbacteria, cell wall components, or culture supernatant in the form of aspray-dried powder is included in or on the surface of the compositiondescribed herein. In one aspect, the isolated Bacillus coagulans is inthe form of a spore. The isolated Bacillus coagulans are at least 85%,at least 90%, at least 95%, or at least 99% pure spores. Alternatively,the isolated Bacillus coagulans is in the form of a vegetative cell. Inone aspect, the isolated Bacillus coagulans are at least 85%, at least90%, or at least 95% pure vegetative cells. In another aspect, theisolated Bacillus coagulans is in the form of a mixture of vegetativecells and spores. The Bacillus coagulans mixture is 90% spores, 10%vegetative cells; 75% spores, 25% vegetative cells; 60% spores, 40%vegetative cells; 50% spores, 50% vegetative cells; 60% vegetativecells, 40% spores; 75% vegetative cells; 25% spores; or 90% vegetativecells, 10% spores.

The Bacillus and/or Bacillus coagulans isolated active agent, e.g.,Bacillus coagulans cell wall and/or Bacillus coagulans culturesupernatant is applied using any of a variety of known methodsincluding, for example, applying a powder, spray-drying the probioticonto the composition, or soaking the composition in a solutioncontaining the probiotic. Optionally, the Bacillus coagulans cell walland/or Bacillus coagulans culture supernatant is dried and reconstitutedin water before use. In another aspect, Bacillus coagulans bacteria inthe form of spray-dried powder administered directly. Optionally, thecomposition comprises about 5×10⁷ CFU Bacillus coagulans bacteria (pergram of composition) in the form of spray-dried powder.

Any of a variety of methods for placing the bacterial composition into acomposition can be used. However, preferred methods include a“spray-dry” method in which the compositions are exposed in a lowhumidity chamber to an atomized mix containing a liquid composition,where the chamber is subsequently exposed to approximately 80-110° F. todry the liquid, thereby impregnating the material of composition withthe components.

A typical concentration is from approximately 1×10⁷ to 1×10¹² CFU; 1×10⁸to 1×10¹¹ CFU; or 1×10⁹ to 1×10¹⁰ CFU of viable bacterium or spores/g ofcomposition. Following drying, the composition is ready for immediateuse or for storage in a sterile package.

The active ingredients (i.e., live bacteria, extracellular components,or cell wall components), comprise between about 0.01% to about 10%;0.01% to about 1%; or about 0.05% to about 0.1% by weight of thecomposition. Optionally, the isolated Bacillus coagulans comprise about1 mg to about 10 g; about 10 mg to about 1 g; or about 25 mg to about 75mg by weight of the composition. Most preferably, the amount of Bacilluscoagulans bacteria is about 5×10⁷ colony forming units (CFU) of bacteriaper gram of composition.

In one aspect, the amount of bacteria is about 10⁴ to 10¹⁴ colonyforming units (CFU) of bacteria per gram of probiotic composition (i.e.,vegetative cells and/or bacterial spores), preferably 10⁵ to 10¹³ CFU/gof composition. Alternatively, the concentrations are 10⁸ to 10¹³ CFU/g;10⁹ to 10¹² CFU/g; or 10¹⁰ to 10¹¹ CFU/g of composition. In one aspect,the amount of bacteria is about 1×10⁶ CFU per gram of composition. Theactual amount in a composition will vary depending upon the amounts ofcomposition to be dispersed into the composition and upon routes ofdispersal.

In one aspect, the invention provides for storing the composition in asterile package at room temperature prior to consumption. Alternatively,the composition is used immediately. In another aspect, the compositioncomprises at least 85%, at least 90%, at least 95% or 100% isolatedBacillus coagulans spores.

By way of example, and not of limitation, Bacillus coagulans spores maybe incorporated into any type of dry or lyophilized product which isdissolved or mixed with hot water, so long as the temperature of theBacillus coagulans mixture is raised to the required heat-shocktemperature (i.e., 80° C. for 5 minutes) necessary for germination ofthe spores. The Bacillus coagulans spores may be incorporated into thedry or lyophilized product by the manufacturer.

In one aspect, the Bacillus coagulans spores survive storage(shelf-life), i.e., retain viability or the ability to germinate atphysiological conditions (e.g., ingestion), from about 12 days to about2 years; from about 1 month to about 18 months; from about 3 months toabout 1 year; or from about 6 months to about 9 months.

Antimicrobial Agents

Optionally, the compositions of the invention also include knownantimicrobial agents, known antiviral agents, known antifungal agents.The other agents in the compositions can be either synergists or activeagents. Preferably, the known antimicrobial, antiviral and/or antifungalagents are probiotic agents compatible with Bacillus coagulans. Thecompositions may also include known antioxidants, buffering agents, andother agents such as coloring agents, flavorings, vitamins or minerals.Thickening agents may be added to the compositions such aspolyvinylpyrrolidone, polyethylene glycol or carboxymethylcellulose.

In one aspect, the active agents are combined with a carrier that isphysiologically compatible with the dermal or epithelial tissue of ahuman or animal to which it is administered. That is, the carrier ispreferably substantially inactive except for surfactant properties usedin making a suspension of the active ingredients. The compositions mayinclude other physiologically active constituents that do not interferewith the efficacy of the active agents in the composition.

A formulated composition of this invention may be completed in weightusing any of a variety of carriers and/or binders. In one aspect,carriers are solid-based dry materials for formulations in tablet,granule or powdered form, and can be liquid or gel-based materials forformulations in liquid or gel forms. Typical carriers for dryformulations include trehalose, malto-dextrin, rice flour,micro-crystalline cellulose (MCC) magnesium stearate, inositol, FOS,gluco-oligosaccharides (GOS), dextrose, sucrose, and the like carriers.Other exemplary composition formulations include a pill, a capsule, or asuspension.

Chemicals used in the present compositions can be obtained from avariety of commercial sources, including Spectrum Quality Products, Inc(Gardena, Calif.), Seltzer Chemicals, Inc., (Carlsbad, Calif.) andJarchem Industries, Inc., (Newark, N.J.).

As described in detail below, the Bacillus coagulans vegetative cells orspores, Bacillus coagulans supernatant, and Bacillus coagulans cell wallcomponents of the invention are useful in enhancement of the immuneresponse. Enhancement of the immune response comprises an increase incytokine (e.g., IL-2, IL-4, IL-6, IL-10, TNF-α, or IFN-γ) production oran increase in immune cell migration to an infection site. Immuneenhancement also includes boosting the immune system by increasingcytokine production, activating the immune surveillance aspect ofpolymorphonuclear leukocytes (PMN), increasing immune cell chemotaxis,activating NK cells, and/or increasing monocyte phagocytosis.

Example 1. Preparation of Bacillus coagulans Cultures

Bacillus coagulans Hammer bacteria (ATCC Accession No. 31284) wasinoculated and grown to a cell density of about 10⁸ to 10⁹ cells/ml innutrient broth containing 5 g Peptone, 3 g Meat extract, 10-30 mg MnSO₄,and 1,000 ml distilled water, adjusted to pH 7.0, using a standardairlift fermentation vessel at 30° C. The range of MnSO₄ acceptable forsporulation is 1 mg/l to 1 g/l. The vegetative cells can activelyreproduce up to 45° C., and the spores are stable up to 90° C. Afterfermentation, the B. coagulans bacterial cells or spores are collectedusing standard methods (e.g., filtration, centrifugation) and thecollected cells and spores can be lyophilized, spray-dried, air-dried orfrozen. As described herein, the supernatant from the cell culture iscollected and used as source of extracellular agents secreted by B.coagulans.

A typical yield from the above culture is in the range of about 10⁹ to10¹⁰ viable spores and more typically about 100 to 150 billioncells/spores per gram before drying. Spores maintain at least 90%viability after drying when stored at room temperature for up to tenyears, and thus the effective shelf life of a composition containing B.coagulans Hammer spores at room temperature is about 10 years.

Example 2. Preparation of Bacillus coagulans Spores

A culture of dried B. coagulans spores was prepared as follows. Tenmillion spores were inoculated into a one liter culture containing 24 gpotato dextrose broth, 10 g of enzymic-digest of poultry and fishtissue, 5 g of FOS and 10 g MnSO₄. The culture was maintained for 72hours under a high oxygen environment at 37° C. to produce culturehaving about 150 billion cells per gram of culture. Thereafter, theculture was filtered to remove culture medium liquid, and the bacterialpellet was re-suspended in water and freeze-dried. The freeze-driedpowder is then ground to a fine powder using standard good manufacturingpractice (GMP).

Example 3. Immunomodulatory and Anti-Inflammatory Effects of BC³⁰ InVitro

Bacillus coagulans (BC) is a gram-positive rod that forms heat-resistantand acid-resistant spores. Oral consumption of spores or encapsulatedspores allows transient colonization of the intestines with BC cultures.The spores germinate and the bacterial cultures grow and ferment thefood in the intestinal lumen.

Immune activation was induced in cultures of human immune cells whenexposed to a) purified Bacillus coagulans culture supernatant (BC1),orb) purified Bacillus coagulans cell wall components (BC2). These twofractions were utilized to characterize the interactions betweenBacillus coagulans and immune cells in vivo, e.g., the Lamina Propria orPeyer's Patches located in the lumen of the intestines. Bacilluscoagulans supernatant (BC1) and Bacillus coagulans cell wall components(BC2) were tested in parallel using a panel of cell-based assays invitro.

Bacillus coagulans was found to positively affect immune cells in thegut: 1) secreted bacterial factors were found to affect/modulate immunecell function; and 2) interaction of bacterial cell wall components withToll-like and/or other immune cell surface receptors led to modulationof immune cell function. BC cell walls contain unique components thatinteract with immune cells in such a way as to activate or boost theimmune system. BC also secretes metabolites and/or other factors thatare produced when BC is growing in the environment of the smallintestine. Such metabolites/factors include, but are not limited toantioxidant and anti-inflammatory compounds.

Bacterial Cell Fractionation

A sample of Bacillus coagulans spores was heat-activated at 50° C. andinoculated in liquid medium. The sample was incubated at 37° C. for 24hours. This time period allows the formation of a log-phase bacterialculture where death and bacterial breakdown is not prominent. After theincubation, the two fractions (Bacillus coagulans supernatant (BC1) andBacillus coagulans cell wall components (BC2)) were prepared. Theinitial separation occurred by decanting the entire culture into a 50 mLvial followed by centrifugation at 2400 rpm. This resulted in thebacteria forming a pellet. The supernatant was gently decanted into anew vial. From this vial, smaller 1 mL samples were aliquoted intoEppendorf vials and subjected to high speed centrifugation, followed bytwo serial filtrations with a 0.2 um filter, to eliminate any intactbacteria and fractions thereof. The sterile, filtered supernatant wasaliquoted and multiple aliquots frozen and stored at −20° C. For laterbiological assays, one aliquot was thawed on each testing day.

The original pellet from the initial centrifugation was used to preparethe cell wall fraction. The wet pellet was frozen and thawed severaltimes to break open the bacterial walls so that the intracellularcompounds could be removed by washing. The thawed slush was transferredto an Eppendorf vial and washed twice in physiological saline using highspeed centrifugation. Then the pellet was transferred to a glass vialand subjected to bead milling using low-protein-binding Zirconium beadswith a diameter of 200 micrometer. The milling was performed by repeated‘pulsing’ using a Vortex mixer. This method is effective to break upcell walls. The beads were removed and the slush containing the brokencell wall fragments were sterile-filtered into multiple aliquots thatwere frozen immediately and stored at −20° C. For later assays, onealiquot was thawed on each testing day.

Purification of Peripheral Blood Mononuclear Cells and PolymorphonuclearCells

Healthy human volunteers between the ages of 20 and 50 years served asblood donors upon informed consent, as approved by the Sky Lakes MedicalCenter Institutional Review Board (FWA 2603). Freshly drawn peripheralvenous blood samples in sodium heparin were layered onto adouble-gradient of Histopaque 1119 and 1077, and centrifuged for 25minutes at 2400 revolutions per minute (rpm). The upper, peripheralblood mononuclear cells (PBMC)-rich and lower polymorphonuclear (PMN)interfaces were harvested using sterile transfer pipettes into newvials, and washed twice with 10 mL phosphate buffered saline (PBS)without calcium or magnesium by centrifugation at 2400 rpm for 10minutes.

Phagocytosis Assay

Evaluation of phagocytic activity was performed using human PMN cells.The choice of particles for phagocytosis was carboxylated Fluorospheres(Molecular Probes, Eugene Oreg.). An aliquot of 0.05 mL Fluorobeads wasremoved from the stock bottle into a 1.5 mL microcentrifuge tube andwashed twice in PBS. Fluorobeads were then re-suspended in 7.5 mL RPMI1640. PMN cells were plated into 96-well plates in RPMI-1640 at aconcentration of 2×10⁶ cells/mL. Ten microliters of 10-fold serialdilutions of BC1 or BC2 were added to test wells in quadruplicate, andPBS was added to control wells in quadruplicate. The plate wasimmediately centrifuged, and the supernatant removed. The cells werere-suspended in RPMI-1640 containing Fluorobeads, and then incubated for2 minutes with Fluorospheres with continuous pipetting. The phagocyticactivity was stopped by adding PBS with 0.02% sodium azide. Cells werewashed twice in PBS with sodium azide to remove beads not ingested bythe cells. Samples were transferred into vials for flow cytometry,ensuring the continued presence of sodium azide. Samples were acquiredby flow cytometry immediately (FacsCalibur, Becton-Dickinson San Jose,Calif.). The analysis was performed using the FlowJo software (TreeStarInc., Ashland OR). During analysis, electronic gating for the PMNpopulation was performed using the forward and side scatter properties.The relative amount of phagocytosis within the PMN population in eachsample was evaluated by the mean fluorescence intensity (MFI) for thegreen fluorescence. The MFI (green) for the untreated samples showed therelative amount of phagocytosis in the absence of BC1 and BC2. The MFI(green) for the BC1 and BC2 treated samples were compared to untreatedsamples.

Reactive Oxygen Species (ROS) Production by PMN Cells

Parallel samples of PMN cells were incubated at 37° C., 5% CO₂ for 20minutes, either untreated or with test products over a range of 10-foldserial dilutions (1:10, 1:100, 1:1000). The precursor dyedichlorofluorescin diacetate (DCF-DA), which becomes brightly greenfluorescent upon exposure to free radicals, was prepared by adding 0.18mL DMSO to a 0.05 mg aliquot of DCF-DA. A working solution of DCF-DA wasthen prepared by adding 0.01 mL stock to 10 mL PBS. The PMN cells werewashed three times in PBS and then re-suspended in the DCF-DA workingsolution and incubated for 1 hour at 37° C. All samples, except for theuntreated control samples, were then exposed to 167 mM H₂O₂ for a periodof 45 minutes to induce ROS production. Samples were washed twice in PBSto remove the peroxide, and transferred to vials for flow cytometry. TheDCF-DA fluorescence intensity in untreated versus H₂O₂-challenged cellswas analyzed by flow cytometry. Data was collected in quadruplicate forcontrols, and duplicate for each dose of BC1 and BC2. The relativeamount of ROS formation in PMN cells was evaluated by green fluorescenceintensity.

PMN Cell Random Migration and Chemotactic Migration Towards ThreeChemo-Attractants: f-MLP, IL-8 and Leukotriene B4

The PMN cell is a highly active and migratory cell type (FIG. 3). Thedifferential effect on PMN cell migration towards the bacterial peptideformyl-Met-Leu-Phe (f-MLP) and two different inflammatorychemo-attractants IL-8 and Leukotriene B4 (LTB4) was tested. Thefollowing experimental model was performed in quadruplicate in order toobtain data significance. Cells were incubated with 10-fold serialdilutions of GBI-30 (GanedenBC³⁰™) supernatant or cell wall fractionsfor 10 minutes in a polystyrene round-bottom tube before platingcommenced. During this time the Millipore trans-well (3.0 μm pore size)migration plate was coated with 50 μg/mL Fibronectin for a period of 30minutes. Chemoattractants and RPMI 1640 were then added to theappropriate bottom chamber wells of the trans-well migration plate in avolume of 150 μL: f-MLP (10 nM), Interleukin-8 (10 μg/mL), andLeukotriene B4 (10 nM). Fibronectin was removed from the top wells byaspiration before plating of cells. Fifty microliters of cells(1×10⁶/mL) were plated in the top chambers, and the top chamber platewas then lowered into the bottom plate and allowed to incubate overnightat 37° C. Quantification of the relative amount of migrated cells wasperformed by fluorescent CyQuant® staining of the cells that hadaccumulated in the bottom chambers. Fluorescence intensity wasquantified in a Tecan Spectrafluor fluorescence plate reader.

Externalization of CD107a on NK Cells in Response to K562 Tumor Cells

Freshly purified human peripheral blood mononuclear cells (PBMC)re-suspended in RPMI 1640 were used for this assay. The cells wereplated at 2×10⁵/well in round-bottom 96-well micro-assay plates, andtreated with serial dilutions of the test products in triplicate.Negative control wells in triplicate were left untreated. In addition, 3wells containing PBMC alone and K562 cells alone served as negativecontrols for baseline CD107a expression. 1×10⁶ K562 cells, an NK-cellsensitive tumor cell line widely used in NK cell cytotoxicity studies,were added to wells containing PBMC with product and untreated PBMC. Thetwo cell types were loosely pelleted by a brief 30-second centrifugationat 2400 rpm followed by incubation at 37° C. for 45 minutes. Cells weretransferred to V-bottom microtiter plates for processing and staining.Cells were stained with CD3-PerCP, CD56-PE and CD107a-FITC. Theexpression of CD107a on the NK cells was determined by flow cytometry.The CD3 negative, CD56 positive NK cells were differentiated from theK562 cells based on forward and side scatter properties, and from otherlymphocytes by electronic gating on CD3⁻, CD56⁺ cells, followed byevaluation of fluorescence intensity for CD107a.

Induction of Natural Killer Cell Activation Markers and Immuno-Staining

Freshly isolated PBMC were plated in a sterile U-bottom 96-well cultureplates (NUNC, Denmark) and treated with serial dilutions of testproducts. For activation of natural killer (NK) and natural killer T(NKT) cells the incubation time was 18 hours. Cells were transferred toV-bottom 96-well plates (NUNC Denmark) and washed in IF buffer (PBScontaining 1% bovine serum albumin and 0.02% sodium azide). Cells werere-suspended in 0.05 mL IF buffer and monoclonal antibodies were addedin previously established optimal quantities (CD3-PerCP, CD56-PE,CD69-FITC, and CD25-FITC: 8 μL/sample), and incubated in the dark atroom temperature for 10 minutes. The cells were washed twice with anadditional 0.15 mL of PBS with 0.02% azide. Following centrifugation andaspiration of the supernatant, the cells were re-suspended in 0.05 mLPBS with 0.02% azide and transferred to 5 mL polystyrene round-bottomtubes each containing 0.4 mL of 1% formalin. Samples were stored in thedark and acquired by flow cytometry within 24 hours using a FACSCaliburflow cytometer (Becton-Dickinson, San Jose Calif.). Analysis wasperformed using the FlowJo (Tree Star Inc., Ashland Oreg.) software.

Modulation of Proliferation and Cytokine Production in Response to PHAand PWM

Freshly purified PBMC re-suspended in RPMI 1640 supplemented with 10%fetal calf serum, L-glutamine (5 mM), penicillin (100 U/mL) andstreptomycin (100 mg/mL) were plated in a U-bottom cell culture plate ata volume of 180 μL at a concentration of 1×10⁶/mL. Next, 20 μL of10-fold serial dilutions of BC1 and BC2 were added to the individualwells in triplicate. In a parallel set of wells, the combinatorialeffect of BC1 and BC2 with known mitogens was tested. Mitogens wereadded at a concentration of 5 μL of PWM (200 μg/mL) and 4 μL of PHA (2μg/mL) to initiate proliferation. The plate was sealed with parafilm andwas incubated at 37° C., 5% CO₂ for 5 days. After 5 days the cells weretransferred to a flat-bottom black 96-well plate and the relative cellnumbers in each culture well quantified by CyQuant® staining and a TecanSpectrafluor fluorescence plate reader.

Cytokine Bead Array

Supernatants from the 5-day lymphocyte proliferation cultures wereharvested and relative levels of the 6 cytokines: IL-2, IL-4, IL-6,IL-10, TNF-α, and IFN-γ were measured using a flow cytometry-based beadarray kit (CBA human Th1/Th2 cytokine kit II, BD Biosciences, San Jose,Calif.). Samples were tested in duplicate according to themanufacturer's specifications, and data acquired immediately by flowcytometry, using a FacsCalibur flow cytometer (Becton-Dickinson SanJose, Calif.). The analysis was performed using the FlowJo software(TreeStar Inc., Ashland, Oreg.).

Statistical Analysis

Statistical analysis involved simple comparisons between two meanvalues, and was performed using Microsoft Excel. Statisticalsignificance was tested using Student's t-test with a p value of lessthan 0.05 indicating a statistically significant difference between twodata sets.

Anti-Bacterial Defense Mechanisms

As described in detail below, Bacillus coagulans supernatant inducedphagocytosis at the highest dose tested, in both PMN(polymorphonuclear—white blood cell (WBC)) cells and monocytes (FIGS.30A and 30B). Both Bacillus coagulans supernatant (BC1) and Bacilluscoagulans cell wall components (BC2) induced random PMN cell migration,i.e., their scavenging activity for invading bacteria (part of normalimmune surveillance). Both fractions, but especially the supernatant,induced PMN cell migration towards a bacterial peptide f-MLP, indicatingthat Bacillus coagulans GanedenBC30™ enhanced the PMN “attack” when abacterial invasion was mimicked. Both fractions, but especially thesupernatant, also enhanced the PMN migration when the Bacillus coagulansGanedenBC30™ fractions were mixed together with f-MLP, i.e., to simulatean in vivo situation in which Bacillus coagulans GanedenBC30™ co-existswith potentially pathogenic bacteria in the gut lumen.

Anti-Viral and Anti-Cancer Defense Mechanisms

NK cells are important in the defense against cancer cells and viruses.Both Bacillus coagulans supernatant (BC1) and Bacillus coagulans cellwall components (BC2) activated Natural Killer (NK) cells. Bothfractions enhanced the aggressive secretion of killer substances from NKcells when the NK cells subsequently were contacted with tumor cells.

Anti-Inflammatory Effects

Bacillus coagulans supernatant (BC1) and Bacillus coagulans cell wallcomponents (BC2) have anti-inflammatory effects. Bacillus coagulansGBI-30 (GanedenBC30™) fractions were introduced to PMN cells at very lowdoses. The PMN cells were then coached to migrate towards aninflammatory mediator, Leukotriene B4. This assay mimics the PMN cell'srole in maintaining an inflammatory cascade. Ganeden BC30™ supports orinhibits the migration of the inflammatory PMN cells, depending onwhether the PMN cell is engaged in normal immune surveillance or isengaged in an inflammatory response.

Example 4. Effect of Bacillus coagulans on Activation of Phagocytes

For each of the figures described in Example 4, the dilutions on theX-axis refers to the tested dilution of each Bacillus coagulans (BC)fraction. For example, a 1:100 dilution is 100-fold dilution of theinitial frozen stock solution.

Reactive Oxygen Species (ROS) Production

Many natural products reduce the reactive oxygen species (ROS) formationin inflammatory cells. However, other products increase ROS formation,indicating an inducement of antimicrobial defense mechanisms. Humanpolymorph nucleated cells (PMN) were used for testing ROS production.This cell type constitutes approximately 70% of the white blood cells inhumans. PMN produce high amounts of ROS upon certain inflammatorystimuli.

Freshly purified PMN were exposed to serial dilutions of the two testproducts, Bacillus coagulans supernatant (BC1) and Bacillus coagulanscell wall components (BC2) in parallel. During the incubation with testproducts, any antioxidant compounds able to cross the cell membrane canenter the interior of the PMN cells. The cells were washed and loadedwith the DCF-DA dye, which fluoresces upon exposure to reactive oxygenspecies. Oxidation was triggered by addition of H₂O₂. The fluorescenceintensity of the PMN cells was evaluated by flow cytometry. The lowfluorescence intensity of untreated control cells served as a baseline,while PMN cells treated with H₂O₂ alone served as a positive control.

If the fluorescence intensity of PMN cells exposed to Bacilluscoagulans, and subsequently exposed to H₂O₂ is reduced compared toexposure to H₂O₂ alone, the test product has anti-inflammatory effects.By contrast, if the fluorescence intensity of PMN cells exposed to atest product is increased compared to H₂O₂ alone, a test product haspro-inflammatory effects.

As shown in FIG. 1, the PMN cell is capable of signaling by both anti-and pro-inflammatory mechanisms, which can lead to either enhancement orreduction of the production of reactive oxygen species (ROS). Both BC1and BC2 showed a clear inhibition of the spontaneous formation ofreactive oxygen species in PMN cells. The effect of BC2 showed adose-dependent inhibition of ROS formation, whereas the effect of BC1showed stronger anti-inflammatory effect at the lowest doses tested. BC1and BC2 presented roughly a 25% inhibition of baseline ROS formation atthe highest dilution of 1:1000. The presence of BC1 (1:1000) reducedspontaneous ROS formation by 22% (P<0.003). BC2 (1:1000) showed asimilar effect on lowering ROS formation (P<0.004). However, at the 1:10dilution of BC2 this effect was even stronger, resulting in a 38%reduction in spontaneous ROS formation (P<0.0002). The inhibition washighly significant (P<0.01) for all dilutions of BC2, and for the 1:1000dilution of BC1. The 1:100 dilution of BC1 was close to being highlysignificant (P=0.0137).

As shown in FIG. 2, when cells were treated with BC1 and BC2 and thenexposed to oxidative stress (H₂O₂), the cells showed a reducedproduction of ROS compared to the negative control. Inhibition remainedat a constant rate of 16-23%. These inhibitions were statisticallysignificant (P<0.05) for both fractions at all dilutions except BC2 at1:1000 (P=0.0539). Both fractions showed highly significant inhibitionof ROS production at the 1:1000 dilution. BC1 (1:100) reduced ROSformation by 23% (P<0.02) while BC2 (1:100) reduced ROS formation by 21%(P<0.008).

Differential Effect on PMN Cell Random Migration and ChemotacticMigration Towards Three Chemo-Attractants: f-MLP, IL-8, and LeukotrieneB4 (LTB-4)

The PMN cell is a highly active and migratory cell type that plays amajor role in immune surveillance. The migratory behavior of PMN isdivided into at least two types: a) random migration and b) directedmigration. Random migration is part of normal immune surveillance, whiledirected migration is migration toward specific chemoattractants.

Both random migration and directed migration were tested in parallel.Directed migration toward the following three distinctly differentchemotactic compounds was examined: i) bacterial peptide f-Met-Leu-Phe(fMLP); ii) the inflammatory cytokine Interleukin-8 (IL-8); and iii)Leukotriene B4 (LTB4).

Some test products may specifically reduce directed PMN migrationtowards the inflammatory mediators IL-8 and/or LTB4, while allowing PMNmigration toward bacterial peptides as part of the normal anti-bacterialimmune defense. The testing of migration towards several inflammatorychemo-attractants helps identify selective responses in this in vitrosystem, which closely mimics the rat paw edema in vivo model ofinflammation. The assay allows for the differentiation betweenantibacterial immune defense mechanisms and inflammation responsemechanisms.

Freshly purified PMN cells were cultured in double-chamber migrationplates, the bottom chamber mimicking tissue, and the top chambermimicking the blood stream (FIG. 3). Cells were plated in the topchambers with or without test products, while different chemoattractantswere placed in the bottom chambers. For control wells, cells were placedin the top chamber without a test product and chemo-attractant was notplaced in the bottom wells. In this manner, evaluation of baselinerandom migration was determined. All assays were performed intriplicate, and repeated at least 3 times with consistent results usingfreshly isolated cells from three different healthy human donors.

Bacillus coagulans supernatant (BC1) was tested at one extra dilution,compared to Bacillus coagulans cell wall components (BC2). Bothfractions of the GanedenBC³⁰™ (Bacillus coagulans) induced the randommigration of PMN cells, indicating that both Bacillus coagulanssupernatant (BC1) and Bacillus coagulans cell wall components (BC2)activated the immune surveillance aspect of PMN cells (FIG. 4). BC1(1:10) increased the random migration by 300% (p<0.001), and BC2 (1:10)increased the random migration by 200% (p<0.005). Higher doses of bothBC1 and BC2 increased the migration towards the bacterial peptide f-MLP,indicating that Bacillus coagulans supernatant (BC1) and Bacilluscoagulans cell wall components (BC2) induced anti-bacterial defensemechanisms (FIG. 5).

Surprisingly, at more dilute concentrations, BC1 and BC2 decreased theamount of f-MLP directed migration, indicating that different levels ofBacillus coagulans have different effects on immune cells in the gut.BC1 (1:10,000) decreased f-MLP directed migration by 11%; however, thiswas not statistically significant, and BC (1:1000) decreased f-MLPdirected migration by 46% (P<0.005).

Treatment of PMN cells with higher doses of BC1 and BC2 enhanced IL-8directed migration (FIG. 6A). This increased migration was highlysignificant at the 1:10 dilution for both BC1 (P<0.002) and BC2(P<0.002). By contrast, lower doses of BC1 and BC2 reduced the IL-8directed migration. At the 1:1000 dilution of BC2, a 65% decrease inmigration was seen that was highly statistically significant(P<0.00001).

A dose study of IL-8 directed PMN migration with much lower doses ofboth BC1 and BC2 was performed. As shown in FIG. 6B, a reduction of inIL-8 directed PMN migration was demonstrated at all dilutions of BC2.This effect of BC2 was strongest at the 10¹⁰ dilution, where migrationwas inhibited by 63% (p<0.02). BC1 treatment of PMN cells at low dosesalso reduced IL-8 directed migration. An interesting pattern of IL-8directed PMN migration inhibition was seen with both BC1 and BC2.Neither product demonstrated a linear dose curve but rather intermediatedoses (10⁴ to 10⁸) of both BC1 and BC2 showed less inhibition of IL-8directed migration compared to higher or lower doses. These resultsindicate that pro- and anti-inflammatory compounds likely co-exist inboth Bacillus coagulans supernatant (BC1) and Bacillus coagulans cellwall components (BC2).

Both BC1 and BC2 fractions also had a dual effect on LTB4 directed PMNmigration. At lower doses, both BC1 and BC2 demonstrated adose-dependent reduction in PMN migration towards LTB4 (FIG. 7). The1:1000 dilution of BC2 inhibited migration by 52% (p<0.002). Cellstreated with 1:1000 (p<0.008) and 1:10000 (p<0.002) dilutions of BC1also showed anti-inflammatory effect that were highly statisticallysignificant. Conversely, the 1:10 dilution of BC1 resulted in asignificant increase in PMN migration towards LTB4 (p<0.003).

Chemoattractant Effect on PMN Cell Migration

The synergistic effect of the following three chemoattractants wasexamined: bacterial f-MLP, IL-8, and Leukotriene B4 (FIG. 8). This assaytests the migratory properties of PMN cells, using the same set-up asdescribed above; however, the BC fractions were not applied to the topchamber with the cells, but were plated in the bottom chamber, therebyproviding a chemotactic gradient. If the test products containedchemoattractant compounds, then the PMN cell migration from the top tothe bottom chamber was increased compared to untreated wells.

In one group, the BC fractions were added to the bottom chambers anddirect chemoattractant properties of the BC fractions were measured. Inanother group, the BC fractions were placed in the bottom chambers incombination with each of the following chemoattractants: bacterialf-MLP, IL-8 and Leukotriene B4 in order to examine the synergisticaffects on PMN migration in the presence of both the BC fractions and aknown chemoattractant.

The two Bacillus coagulans fractions have varying effects despite comingfrom the same bacterial cultures (FIG. 8). Higher doses of Bacilluscoagulans supernatant (BC1) had a strong chemotactic effect, whileBacillus coagulans cell wall components (BC2) did not show anychemoattractant effect, even at the highest dose. By contrast, BC1decreased the amount of migration, indicating that BC1 has ananti-inflammatory effect.

As shown in FIG. 9, the two Bacillus coagulans fractions had verydifferent effects on the f-MLP directed migration. Higher doses of BC1enhanced the f-MLP directed migratory activity. The chemoattractanteffect of BC1 (1:10) increased migration by 55% (P<0.01). With theexception of the highest dose of BC2, this fraction reduced migrationtowards f-MLP. The interaction of PMN cells with BC2 made the cells muchless responsive to f-MLP. BC2 (1:1000) treated cells inhibited migrationby 52% (P<0.001).

As shown in FIG. 10, the two Bacillus coagulans fractions also had verydifferent effects on the IL-8 directed migration. All doses of BC1enhanced the IL-8 directed migratory activity. The chemoattractanteffect of BC1 (1:10) was statistically significant (P<0.00001). BC2 hada dual effect. At higher doses BC2 enhanced IL-8 induced migration.However, at the lowest dose tested, this fraction reduced migrationtowards IL-8. The interaction of PMN cells with BC2 made the cells muchless responsive to IL-8. BC2 (1:1000) treated cells inhibited migrationby 49% (P<0.004).

As shown in FIG. 11, the two Bacillus coagulans fractions also had verydifferent effects on the LTB4 directed migration. All doses of BC1enhanced the LTB4 directed migratory activity. The chemoattractanteffect of BC1 (1:10) was statistically significant (P<0.002).

BC2 had a dual effect. At the higher dose, it enhanced LTB4 inducedmigration. However, at the 1:100 dose, this fraction reduced migrationtowards LTB4. The interaction of PMN cells with BC2 made the cells lessresponsive to LTB4. BC2 (1:100) treated cells inhibited migration by 11%(P<0.01).

Effect on Macrophage Phagocytic Activity

Phagocytosis of microbial particles is an important part of the innateimmune response. It is a rapid process, and the effect of a test producton enhancing this cellular function can be almost immediate.Phagocytosis was measured by how well PMN cells engulfed greenfluorescent carboxylated fluorospheres. The mean fluorescence intensity(MFI) of phagocytic cells was then evaluated by flow cytometry. Freshlypurified peripheral blood mononuclear cells were pretreated with eitherBacillus coagulans supernatant (BC1) or Bacillus coagulans cell wallcomponents (BC2) for 3 minutes, and then introduced to fluorescentmicro-particles mimicking bacteria. The cells were allowed to ingestparticles for 2 minutes, after which free micro-particles were removedby centrifugation. The fluorescence intensity of the phagocytes was thenevaluated by flow cytometry. Electronic gating was performed on themonocyte population, and the analysis was performed by measuring themean fluorescence intensity (MFI FL3). A faster or stronger rate ofphagocytosis results in a higher number of fluorescent micro-particlesper cell.

As shown in FIG. 12, BC1 at the dilution of 1:10 increased monocytephagocytosis. This increase of 28% was highly significant (P<0.01). Asshown in FIG. 13, BC1 and BC2 increased phagocytosis in PMN cellscompared to the negative controls. Exposure of PMN cells to BC1 at the1:10 dilution increased phagocytosis by 40% (p<0.02). Exposure of PMNcells to BC2 at the 1:10 dilution increased phagocytosis by 25%(p<0.008). Further dilutions of both products resulted in reduced PMNphagocytosis (p<0.05).

The images in FIGS. 30A and 30B represent photographs taken from afluorescence microscope. The control shows an untreated PMN cell thathas engaged in phagocytosis (FIG. 30A). The green beads are carboxylatedfluorospheres that mimic bacterial particles. FIG. 30B shows a PMN cellthat has been treated with BC2. This cell has ingested many morefluorospheres than the untreated cell in FIG. 30A. This figure, incombination with the data above, indicates that Bacillus coagulansincreases the capacity of phagocytes for engulfing foreign material.

Example 5. Activation of NK Cells

Natural Killer (NK) cells are involved in the primary defense mechanismsagainst transformed cells and viruses. These cells travel in the bloodstream in a state of rest, but can be immediately activated to a) killcancer cells by either cell contact or secretion of cytotoxic compoundssuch as perforin and granzyme, b) proliferate, and c) secrete substancesthat attract other cells into the site. In order to investigate apossible effect of BC1 and BC2 on NK cell activation, the changes inexpression of the NK activation cell surface marker CD69 were examined.The increased expression of this marker has been associated with anincreased cytotoxic activity of NK cells (Clausen et al, 2003Immunobiol, 207(2):85-93).

Freshly purified human peripheral blood mononuclear cells were used forthese assays. The cells were plated in 96-well micro-assay plates intriplicate. Negative control wells in triplicate were left untreated.Positive controls were treated with IL-2 at a dose of 100 internationalunits per mL (IU/mL). After 18 hours of culture, cells were stained forthe activation molecule CD69 and the growth factor receptor CD25 on thesurface of CD3-negative, CD56-positive NK cells, and on CD3-positive,CD56-positive NKT lymphocytes to evaluate activation of NK and/or NKTcells in vitro.

FIG. 14 depicts the change in mean fluorescence intensity of the NKactivation marker CD69, following exposure of NK cells to eitherBacillus coagulans supernatant (BC1) or Bacillus coagulans cell wallcomponents (BC2). Both BC1 and BC2 showed a clear dose-dependentinduction of the expression of CD69 on NK cells. The effect reached highstatistical significance for both BC1 and BC2 at a 1:400 dilution, whereCD69 expression was increased by 32% (p<0.01) for BC1, and by 36% forBC2 (p<0.003).

FIG. 15 shows the expression of CD25 on NKT cells. For both BC1 and BC2,there was not a large difference in CD25 expression compared to baselinelevels. While the 1:100 dilution of BC1 resulted in an increase inexpression, this change did not reach statistical significance. Nochanges in CD25 expression on T cells were observed when comparinguntreated cells to those treated with either BC1 or BC2.

Externalization of CD107a on NK Cells in Response to Tumor Cells

One of the functions of NK cells is to kill tumor cells andvirus-infected cells via cell-cell contact and by secretion ofsubstances such as Perform During this process, the CD107a receptorexpressed on the interior of granules in the cytoplasm of NK cells istransiently brought to the cell surface. Thus, CD107a expression on NKcells is a measure of their cytotoxic activity by secretion of cytotoxicsubstances.

Freshly-purified human peripheral blood mononuclear cells were used forthis assay. The cells were plated in round-bottom 96-well micro-assayplates, and treated with serial dilutions of the test products intriplicate. Negative control wells in triplicate were left untreated.All other wells were used for addition of the NK-cell sensitive K562tumor cell line, widely used in NK cell cytotoxicity assays. Positivecontrol wells were left without adding test products. All remainingwells were treated with serial dilutions of test products. The two celltypes were brought physically together by a brief 15-secondscentrifugation, and incubated for 45 minutes at 37° C. Cells weretransferred to V-bottom microtiter plates for processing and staining.The expression of CD107a on the NK cells was analyzed by flow cytometry,where the NK cells were differentiated from the other lymphocytes basedon positive staining for CD3 and CD56, and from the K562 cells based onsize. Cytotoxic activity of NK cells in vitro was measured. The responsein this assay predicts a similar response to non-malignant, virallyinfected cells.

FIG. 16 shows the change in mean fluorescence intensity (MFI) of CD107aexpression on natural killer cells that have been exposed to tumorcells, with or without the addition of BC1 or BC2. Both BC1 and BC2 showa mild increase in CD107a cell surface expression, with BC2 having thestrongest effect at the 1:200 dilution; the effect did not quite reachstatistical significance (p<0.07).

Example 6. Support of Adaptive Immune Function: Modulation of LymphocyteProliferation and Cytokine Production in Response to Two Known Mitogens

A series of assays were performed to determine if the test productswould trigger exaggerated immune reactions. As part of a standard safetytesting of natural products, test products were examined for mitogenicpotential, that is, whether they induce cell division in healthy humanlymphocytes. Simultaneous to the test of mitogenic potential, the testproducts were examined for their effect on cells responsible for theadaptive immune defense, that is, T and B lymphocytes. The lymphocyteproliferation assay offers a simple method to assess whether thecompositions alter lymphocyte responsiveness to known signals. A changein the proliferative response to known mitogens in the presence of acomposition indicates an immunomodulatory effect, such as T and Blymphocyte signaling and activation.

The compositions were tested in serial dilutions in the presence andabsence of mitogens. Two mitogens were tested in parallel:Phytohemagglutinin (PHA), which is a T cell mitogen that will induce Tcell proliferation, and Pokeweed Mitogen (PWM), which is a mitogen thatrequires the collaboration of T cells, B cells and monocytes in theculture. The PHA is a cleaner signal, but the PWM is a morephysiological signal.

Freshly purified human peripheral blood mononuclear cells (PBMC) werecultured in the absence versus presence of serial dilutions of thecompositions. Three parallel sets of cultures were established, whereone tested the direct effect of test product on lymphocyteproliferation, and the two others examined the interference of thecomposition with response to the known mitogens. Positive controlsincluded cells treated only with a mitogen in the absence of testproduct. A change (increase, decrease) of mitogen-induced proliferationis a strong indication of the presence of immunomodulating compounds.

Neither BC1 nor BC2 had a mitogenic effect on lymphocyte proliferationfollowing five days incubation at 37° C. with product and culture media.

Both BC1 and BC2 showed a reduction in lymphocyte proliferation in thepresence of PHA and PWM (FIGS. 17 and 18). This reduction wassignificant at all doses of BC1 in the presence of both PHA and PWM(p<0.02) and was significant for the two highest concentrations of BC2(p<0.02). Additionally, high statistical significance was reached forBC1 at the 1:10 (p<0.003) and 1:100 (p<0.002) doses and BC2 at the 1:10(p<0.004) dose in the presence of PHA and BC1 at the 1:100 (p<0.005)dose and BC2 at the 1:10 (p<0.002) and 1:100 (p<0.006) doses in thepresence of PWM.

Cytometric Bead Array

A flow cytometry-based Th1/Th2 cytokine bead array (CBA) for the 6cytokines IL-2, IL-4, IL-6, IL-10, TNF-α and INF-γ was used to evaluatethe levels of cytokines present in the supernatants from 5-daylymphocyte cultures. In FIGS. 19-20, relative changes in cytokineconcentrations are first presented in overview graphs showing changes inall 6 cytokines across the 3 product dilutions. There are separategraphs for BC1 and BC2 and changes are represented as factor of changefrom baseline (lymphocytes cultured without product).

Lymphocytes were also cultured (with or without product) in the presenceof 2 different mitogens. Phytohemagglutinin (PHA) was used to induce Tcell proliferation, and Pokeweed Mitogen (PWM) was used to induce T andB lymphocyte proliferation in a process that requires the collaborationof T cells, B cells, and monocytes in the culture. Comparisons were madebetween lymphocytes cultured in the presence of no product andlymphocytes cultured in the presence of 1:100 dilutions of either BC1 orBC2. This data is presented in separate graphs for each individualcytokine and also compares changes in cytokine levels in lymphocytesthat were cultured without mitogens either without product or with the1:100 dilutions of BC1 and BC2.

In the absence of mitogens, both BC1 and BC2 treatment of PBMC led todecreased IL-2 levels compared to untreated PBMC (FIG. 21). Thisreduction was statistically significant for BC1 and BC2 (p<0.002). Nostatistically significant changes in IL-2 levels were observed with BC1or BC2 treatment in the presence of either mitogen, compared to BC1versus BC2 treatment alone.

In the absence of mitogens, both BC1 and BC2 treatment of PBMC led toincreased IL-4 levels compared to untreated PBMC (FIG. 22). Thisincrease was statistically significant for both BC1 (p<0.002) and BC2(p<0.01). No statistically significant changes in IL-4 levels wereobserved with BC1 or BC2 treatment in the presence of either mitogen,compared to BC1 versus BC2 treatment alone.

As shown in FIG. 23, both BC1 and BC2 treatment of PBMC, in the absenceof mitogens, led to massive induction of IL-6 production. The increasewas highly statistically significant (P<0.00002). No statisticallysignificant changes in IL-6 levels were observed with BC1 or BC2treatment in the presence of Pokeweed mitogen, compared to BC1 versusBC2 treatment alone. The IL-6 induction by both BC1 (P<0.001) and BC2(P<0.0009) was found to be highly statistically significant.

As shown in FIG. 24, both BC1 and BC2 treatment of PBMC, in the absenceof mitogens, led to induction of IL-10 production. The increase washighly significant (P<0.008). PBMC treated with both BC1 and PHA led tohigher IL-10 production (P<0.0009) than if cells were treated witheither product alone. Treatment of PBMC with BC1 and PWM also led to anincrease in IL-10 production; however, the data was not found to bestatistically significant. No statistically significant changes in IL-6levels were observed with BC2 treatment in the presence of eithermitogen when compared to BC2 treatment alone.

In the absence of mitogens, TNF-α production was slightly lower thanuntreated PBMC in the presence of both BC1 and BC2 (FIGS. 25A and 25B).This mild reduction was not statistically significant for either BC1 orBC2. Treatment of PBMC with either BC1 or BC2 in the presence of PHAresulted in 2-fold decreases in TNF-α expression that were statisticallysignificant for both BC1 (P<0.002) and BC2 (P<0.006). In contrast,treatment of PBMC with BC1 and BC2 in the presence of PWM resulted instrong increases in TNF-α levels. In the presence of PWM, BC1 treatmentproduced an 11-fold increase (P<0.003) and BC2 treatment a 22-foldincrease (P<0.001).

In the absence of mitogens, INF-γ levels increased in response totreatment with both BC1 and BC2 (FIGS. 26A and 26B). These changes werehighly statistically significant for both BC1 (P<0.001) and BC2(P<0.0004). Treatment of PBMC with either BC1 or BC2 in the presence ofPHA did not produce statistically significant changes in INF-γexpression. In contrast, treatment of PBMC with BC1 and BC2 in thepresence of PWM resulted in 3-fold (BC1) and 4-fold (BC2) increases inINF-γ levels, both of which were statistically significant (P<0.0004).

Example 7. Anti-Oxidant Effects: Cell-Based Antioxidant Protection Assay

The culture supernatant and cell wall fractions were tested in theCell-based Antioxidant Protection in Erythrocytes (CAP-e) assay, abioassay for antioxidants test (FIGS. 27-29). This assay allowsassessment of antioxidant potential in a method that is comparable tothe oxygen radical absorbance capacity (ORAC) test, but only allowsmeasurement of anti-oxidants that are able to cross the lipid bilayercell membrane. As a model cell type, red blood cells (RBC) were used.This is an inert cell type, in contrast to other cell types such as PMNcells, where pro-inflammatory compounds may induce the reactiveoxidative burst. This assay is particularly useful to assessantioxidants from complex natural products in a cell-based system.

Freshly purified human RBC were washed repeatedly in physiologicalsaline, and then exposed to the test compositions. During the incubationwith a test product, any antioxidant compounds able to cross the cellmembrane can enter the interior of the RBC. RBC were then washed toremove compounds that were not absorbed by the cells, and loaded withthe DCF-DA dye, which turns fluorescent upon exposure to reactive oxygenspecies. Oxidation was triggered by addition of the peroxyl free radicalgenerator AAPH. The fluorescence intensity was evaluated. The lowfluorescence intensity of untreated control cells serve as a baseline,and RBC treated with AAPH alone serve as a positive control for maximumoxidative damage. An observation of a reduced fluorescence intensity ofRBC exposed to a test product and subsequently exposed to AAPH,indicates that the test product contains antioxidants available topenetrate into the cells and protect these from oxidative damage.

For the testing of BC1 and BC2, the antioxidant protection capacity inboth freshly isolated RBC and in RBC that had been stored for 45 dayswas tested. Fresh RBC contain antioxidants derived from food as well asredox enzymes. With storage, the antioxidants get depleted, and theenzymatic functions may decline over time. Both BC1 and BC2 containedcompounds that could enter into RBC. These compounds entered moreefficiently into fresh RBC. However, the compounds from BC1 possessed noantioxidant capacity. In contrast, they interfered with the antioxidantprotective mechanisms inside the RBC. A very mild antioxidant protectionis seen in aged cells exposed to BC2.

Modulation of Immune Responses by Cell-Free BC Supernatants (BC-1) andCell-Free Cell Wall Fractions (BC-2)

In summary, both BC1 and BC2 inhibited spontaneous ROS formation andreduced ROS formation when oxidative stress had been applied to PMN's.At higher concentrations, both BC1 and BC2 increased the migration ofPMN cells towards a bacterial peptide, indicating an enhancement of theimmune surveillance function of PMN in detecting bacteria. Conversely,at low concentrations, both BC1 and BC2 decreased the migration of PMNcells towards a bacterial peptide, indicating that at lowconcentrations, the compounds present in the supernatant and cell wallpreparations of Bacillus coagulans had an immunomodulatory effect on theability of the PMN to respond to the bacterial peptide signal. Thiseffect underlies processes that dictate how the immune system respondsto a bacterial infection vs. resident (beneficial) commensal bacteria inthe gut. The highest dose of BC1 increased monocyte phagocytosis. BC2did not increase monocyte phagocytosis. BC1 and BC2 at the strongestdose increased PMN phagocytosis. NK expression (CD69) was increased inall dilutions of BC1 and BC2 tested. BC1 and BC2 both deceasedlymphocyte proliferation at all doses. Both Bacillus coagulans fractionsreduced IL-2 and TNF. These cytokines are known TH1 cytokines which isdirected at macrophage activation. However, a mild increase was noticedfor IFN-γ production for both Bacillus coagulans fractions. BothBacillus coagulans fractions increased the production of IL-4, IL-6, andIL-10. These cytokines are more directly linked to TH2 production whichis used to help signal and activate B-cells, which are antigenpresenting cells for the adaptive immune system. BC1 and BC2 were bothcapable at hindering the migration of PMN cells when directed towardsthe known chemoattractant IL-8. This strong anti-inflammatory effect wassignificant across a wide range of dilutions. Bacillus coagulans cellwall inhibited the migration of PMN cells when directed towards theinflammatory chemoattractant LTB4.

Example 9: Fractionation of Bacillus coagulans Supernatant and Cell WallComponents

Different molecular weight compounds in Bacillus coagulans supernatantand cell wall components were fractionated/purified to evaluate theirbiological effects. Three molecular range fractions are examined forvarious biological activities.

Preparation of the Two Test Fractions (Cell Wall and Supernatant) andSpores.

A sample of BC spores was heat-activated and inoculated in a liquidculture medium. Sample is incubated at 37° C. for 24 hours. This timeperiod allows the formation of a log-phase bacterial culture where deathand bacterial breakdown is not prominent. After the incubation, the twofractions (cell wall and supernatant) are prepared. The initialseparation is accomplished by decanting the entire culture into a 50 mLvial and centrifugation at 2400 rpm. Bacterial aggregates form a pellet.The supernatant is gently be decanted into a new vial. From this vial,smaller 1 mL samples are aliquoted into Eppendorf vials and subjected tohigh speed centrifugation, followed by three serial filtrations, toeliminate any intact bacteria and fractions thereof. The sterile,filtered supernatant is aliquoted and multiple aliquots frozenimmediately at −80° C. The original pellet from the initialcentrifugation is used to prepare the cell wall fraction. The wet pelletis frozen and thawed. The thawed slush is transferred to an Eppendorfvial and washed twice in physiological saline using high speedcentrifugation. Then the pellet is transferred to a glass vial andsubjected to bead milling using low-protein-binding Zirconium beads witha diameter of 100 micrometer. The milling is performed by repeated‘pulsing’ using a Vortex mixer. The beads are removed and the slushcontaining the broken cell wall fragments are sterile-filtered intomultiple aliquots that will be frozen immediately at −80° C.

Biological Activity of Bacillus coagulans Supernatant and Cell WallComponents

In order to identify the molecular weights of predominantprotein/carbohydrate compounds in the supernatant and cell wallfractions of BC, Electrophoresis is used to understand the protein andpolysaccharide makeup of Bacillus coagulans fractions and spores. Atypical protein gel electrophoresis method is shown in FIG. 31. Thisprocess separates the proteins and polysaccharides by molecular weightand gives a valuable fingerprint for each of the BC fractions.Electrophoretic separation provides information about the relativequantity of specific proteins and polysaccharides in the product.

Gel electrophoresis of the previous batch of supernatant and cell wallfractions showed several regions of interest. The supernatant containscompounds lower than 5-10 kDa, i.e., lower than the range that can beclearly fractionated by SDS gelelectrophoresis (see, smear below thewords “BC Supernatant” in FIG. 32). Both fractions contained doublebands in the 10 kDa range. The supernatant contained several additionalprominent bands between 20-30 kDa and between 50-150 kDa. Fractionationof the supernatant and the cell wall fraction is carried out to yieldthree fractions or purified preparations A) Below 3 kDa, B) between 3-30kDa, and purification C) between 30-200 kDa. The major bioactivecompounds from the cell wall are in fraction B. Electrophoresis is usedas a tool to ensure product consistency during stages of productdevelopment. It is also useful as a regular quality control tool duringmanufacturing.

Size fractionation by molecular weight (<3, 3-30, 30-200 kDa) of bothsupernatant and cell wall fractions is performed to further characterizethe three main identified biological activities: a) Anti-inflammatoryeffect, as measured by inhibition of cell migration in response toinflammatory mediators; b) Effect on NK cell activation; and c) Effecton cytokine production.

Anti-Inflammatory Effect: Inhibition of Leukotriene B4 DirectedMigration

The PMN cell is a highly active and migratory cell type. Bacilluscoagulans fractions have strong anti-inflammatory effects when exposedto the known inflammatory cytokine LTB4. Crude BC cell wall and BCsupernatant are fractionated into the following molecular weight ranges:a)<3 kDa, b) 3-30 kDa, and c) 30-200 kDa. Similar volumes of Bacilluscoagulans cell wall and supernatant are placed into centrifugationcolumns that filter out specific molecular weight fractions. Aftercentrifugation the remaining volumes are serial diluted and placed inwith the PMN's before plating into the top chamber.

Freshly purified PMN cells cultures are set up in double-chambermigration plates, where the bottom chamber mimics tissue, and the topchamber mimics the blood stream as described in FIG. 33. Cells areplated in the top chambers with and without test products, and thedifferent chemo-attractant (LTB4) is present in the bottom chambers. Allassays are performed in triplicates, and repeated at least 3 times withconsistent results. The testing of migration towards the inflammatorychemo-attractant LTB4 identifies selective responses in this in vitrosystem, which closely mimics some in vivo models of inflammation, suchas rat paw edema. The assay allows a distinction between normal PMNdefense mechanisms versus response to inflammation. Theanti-inflammatory activity of Bacillus coagulans spores is alsoexamined. These assays identify which molecular weight compounds areresponsible for the anti-inflammatory effects of BC supernatant and cellwall fractions.

Natural Killer Cell Activation (CD69 Expression)

Crude BC cell wall and BC supernatant are fractionated into thefollowing molecular weight ranges: a)<3 kDa, b) 3-30 kDa, and c) 30-200kDa. As described above, both BC fractions activated NK cells. Inductionof the CD69 activation marker on the NK cells is determined. Freshlypurified human peripheral blood mononuclear cells are used for theseassays. The cells are plated in 96-well micro-assay plates intriplicate. Negative control wells in triplicate are left untreated.Positive controls are treated with IL-2 at a dose of 100 internationalunits per mL (IU/mL). After 18 hours of culture, cells are stained forthe activation molecule CD69 on the surface of CD3-negative,CD56-positive NK cells.

Biological activity of Bacillus coagulans supernatant and cell wallcomponents is also assessed after drying and reconstitution to determineif bioactivity is preserved after drying.

This assay identifies which molecular weight compounds are responsiblefor the NK cell activating effects of BC supernatant and cell wallfractions. The ability of Bacillus coagulans spores to activate NK cellsis also examined.

Cytokine Production

Crude BC cell wall and BC supernatant are fractionated into thefollowing molecular weight ranges: a) <3 kDa, b) 3-30 kDa, and c) 30-200kDa. Previous experiments showed that the BC fractions directly inducedchanges in cytokine production. The fractions will be examined toidentify which molecular weight ranges of compounds are responsible forthis change in the BC supernatant and cell wall fractions. Freshlypurified human peripheral blood mononuclear cells (PBMC) will becultured in the absence versus presence of serial dilutions of BCfractions. Biological activity of Bacillus coagulans supernatant andcell wall components is also assessed after drying and reconstitution todetermine if bioactivity is preserved after drying.

The ability of Bacillus coagulans spores to induce the production ofcytokines IL-2, IL-4, IL-6, IL-10, TNF-alpha, and IFN-gamma is alsoexamined.

Example 10: A Controlled Trial to Evaluate the Effects of GBI-30(GanedenBC³⁰) (Viable Cells and Spores) on the Immune System

The beneficial effect of GanedenBC³⁰ (Bacillus coagulans GBI-30, ATCCDesignation Number PTA-6086) on the immune system in healthy individualswas evaluated when challenged with the adenovirus and influenza. Studieswere also carried out to determine the beneficial effect of GanedenBC³⁰(Bacillus coagulans GBI-30, ATCC Designation Number PTA-6086) in healthindividuals on % CD3CD69 cells—a marker for T-lymphocyte activity.

Ten healthy adult subjects were recruited for this study. No concurrentillness or recent immunization was allowed. Blood was drawn at baselineat day 0. Subjects were instructed to consume 1 capsule daily containing500 million CFU's of GanedenBC³⁰ daily for 30 days. Blood was drawnagain on day 30. Due to one subject being statistically different atbaseline, only 9 subjects were used in the final analysis.

Blood samples were taken and stimulated with either the adenovirus orinfluenza A and incubated for 24 hours and then vortexed. 100microliters of the sample was drawn off and of that 20 microliters wereused for the % CD3CD69 testing. 900 microliters from the remainingsamples were drawn and centrifuged and the plasma removed. Samples ofthe plasma were taken and used in the cytokine testing. Variouscytokines were tested, and ones with statistically changes are notedbelow.

When 500 million CFU/day were consumed, the immune system of subjectswas boosted when challenged both with the adenovirus and influenza A.The % CD9CD69 cells, were increased after demonstrating an ability toincrease T-lymphocyte activity. Statistically significant changes wereseen in IL-8 production (P=0.039) when exposed to influenza A, and INF-γproduction (P=0.039) when exposed to adenovirus. Statisticallysignificant increases in % CD3CD69 (P=0.023) were seen both toadenovirus and influenza A.

These data indicate that a probiotic composition containing 2 billionCFU of GanedenBC³⁰, when consumed daily, boosted the immune system. Whenconsumed at only 500 million CFU/day, the composition was able todemonstrate statistically significant boosts to the immune system aswell as increasing T-lymphocyte activity.

Other embodiments are within the scope and spirit of the invention. Itwill be recognized by a person of ordinary skill in the art that variouscomponents of the examples described herein can be interchanged and/orsubstituted with various components in other examples, and that othermodifications may be possible. To the extent that any of the materialincorporated by reference herein conflicts with the terms of the presentdisclosure, the present disclosure is intended to be controlling.Further, while the description above refers to the invention, thedescription may include more than one invention.

1.-14. (canceled)
 15. A food or beverage composition comprising purifiedcell wall components of a Bacillus coagulans bacterium, the cell wallcomponents being present in an immune system boosting amount.
 16. Thecomposition of claim 15, wherein the purified cell wall components aredried or lyophilized.
 17. The composition of claim 15, wherein the cellwall components comprise a bioactive compound between 3 and 200 kDa. 18.The composition of claim 15, wherein the cell wall components comprise abioactive compound between 30 and 200 kDa.
 19. The composition of claim15, wherein the cell wall components comprise a bioactive compoundbetween 3 and 30 kDa.
 20. The composition of claim 15, wherein the cellwall components comprise a bioactive compound less than 3 kDa.
 21. Thecomposition of claim 15, further comprising a supernatant of Bacilluscoagulans.
 22. The composition of claim 21, wherein the supernatant isdried supernatant.
 23. The composition of claim 21, wherein thesupernatant comprises a bioactive compound between 30 and 200 kDa. 24.The composition of claim 21, wherein the supernatant comprises abioactive compound between 3 and 30 kDa.
 25. The composition of claim21, wherein the supernatant comprises a bioactive compound less than 3kDa.
 26. The composition of claim 15, wherein the Bacillus coagulanscomprises Bacillus coagulans hammer strain Accession No. ATCC 31284, ora strain derived from Bacillus coagulans hammer strain Accession No.ATCC
 31284. 27. The composition of claim 15, wherein the Bacilluscoagulans comprises GBI-30 strain ATCC Designation Number PTA-6086. 28.The composition of claim 15, wherein the Bacillus coagulans comprisesGBI-20 strain ATCC Designation Number PTA-6085.
 29. The composition ofclaim 15, wherein the Bacillus coagulans comprises GBI-40 strain ATCCDesignation Number PTA-6087.